Background
Acid-sensing ion channels (ASICs) are a group of sodium-selective ion channels activated by extracellular acidosis. They are widely expressed in neurons of both the central and the peripheral nervous system and they have also been detected in nonneuronal tissues [
1‐
3]. The participation of the ASICs in sensory processes, synaptic plasticity, learning and memory, and in the survival of neurons following global ischemia in the brain has been demonstrated in various animal models [
2,
4‐
8]. The role of ASICs in nociception has been shown in different models of pain and inflammatory hyperalgesia [
9,
10]. The cutaneous pain produced in humans by moderate pH (up to 6) is largely mediated by the ASICs [
11,
12] The pain sensation produced by cardiac ischemia seems also to be mediated by ASIC activation [
13].
The search for new selective pharmacological agents with no significant side-effects is an increasing requirement for the development of new drugs to be used in the treatment of acute and chronic pain. The standardized extract named BM-21 obtained from the sea grass
Thalassia testudinum, a marine plant highly abundant along the coasts of Cuba, has been characterized and patented [
14]. This product is rich in flavonoid compounds and it does not possess any known toxicity, but has antiinflammatory and antioxidant properties and contributes to the recovery of irradiation-damaged dermis and the normal properties of the epidermis [
15,
16]. The bioassay-guided fractionation of the plant extract resulted in the isolation of thalassiolin B (chrysoeriol 7-β-D-glucopyranosyl-2"-sulphate), the most abundant component of the extract. Its topical application (240 μg cm
-2) markedly reduced skin UVB-caused damage. Thalassiolin B scavenged the 2,2-diphenyl-2-picrylhydracyl radical (EC50 = 100 μg mL
-1) suggesting that it may contribute to the protective effect on the skin of the crude extract of
T. testudinum
[
17].
In this study we describe the antinociceptive effects of BM-21 and thalassiolin B on different animal models of pain, and the inhibitory action of BM-21 and thalassiolin B on the ASIC currents in isolated dorsal-root ganglion (DRG) neurons in a primary culture.
Discussion
This study demonstrates the antinociceptive action of BM-21 in the classical pharmacological models of thermal- (hot plate test) and chemical-caused (acetic acid and formalin tests) pain in mice and of thalassiolin B in the formalin test. The inhibition of the nociceptive behavior does not seem to result from a nonspecific muscle-relaxant or sedative effect because the BM-21 extract did not show any motor performance alterations in the rota-rod test.
The hot plate test is one of the most commonly used for nociception studies [
22]. The increase in temperature produces two behavioral components (paw licking and jumping) that can be measured by their reaction times. In this model, BM-21 significantly increased the latency for jumping or licking suggesting that the extract exerts an acute antinociceptive action [
23]. The acetic acid-generated writhing test has been used for the assessment of analgesic or antiinflammatory properties of several drugs. Oral administration of BM-21 reduced contortions and stretches thus indicating it is exerting an antinociceptive action in persistent tonic pain.
Nociceptive behavior in the formalin test has been divided into two phases [
22,
24]. The initial 1st-phase (nociceptive pain) is considered to be caused predominantly by activation of C fibers, whereas the 2nd-phase is associated with inflammatory components with release of different pain mediators (22,23,24). Reactive oxygen species (ROS) have been documented to contribute to and maintain conditions of chronic pain [
25]. Both phases are sensitive to centrally acting drugs such as opioids [
26], but the 2nd-phase is also sensitive to corticosteroids and nonsteroidal antiinflammatory drugs (NSAIDs), especially when the formalin is injected in high concentrations [
26‐
29]. Antioxidants attenuate both phases of the responses in mice [
25]. It has been established that COX-1 is involved in the 2nd-phase response in the formalin test and in the writhing test in mice [
30]. A previous report has demonstrated the inhibition in vitro of the activity of both phospholipase A2 and COX-1 by BM-21[
31]. Thus, the dual effect exerted by BM-21 on the arachidonic acid pathway could account for its effect in the 2nd-phase of the formalin test and in the writhing test. The BM-21 and thalassiolin B also have ROS scavenging properties. These could contribute to the antinociceptive effects observed in vivo, however in the DRG experiments in vitro this effect could be minor or absent. The contribution of ASIC channels in the formalin test has been documented and consistently both amiloride and thalassiolin B attenuated painful behavior in the late phase. For amiloride our data show similarities with those reported in the literature [
32]. Albeit it has been found that amiloride use in the formalin test attenuated the 2nd-phase in female, but not in male, CD 1 mice (except at a toxic dose) [
33]. The effect of BM-21 on both the formalin and writhing tests support that its profile is consistent with antiinflammatory analgesic drugs, however a phasic-antinociceptive action is also produced, particularly bearing in mind the antinociceptive effects of BM-21 on the hot plate model and the effect of thalassiolin B on the first phase of the formalin test.
Regalado and coworkers determined that the phenolic content in BM-21 was 18 ± 1.5% [
17]. Consistent with this, the separation procedure of the plant extract resulted in the isolation of thalassiolin B as the major phenolic constituent of the extract. Phenolic compounds, and particularly flavonoids, have a wide variety of biological activities in mammals [
34,
35]. Our results indicate that flavonoids, in particular thalassiolin B, may contribute in a significant degree to the biological action of BM-21. In fact, the studies in vivo using the formalin test in mice support that thalassiolin B per se has an antinociceptive action.
Some mechanisms of nociception are known to involve ASICs [
2]. Previous results have documented that they sense extracellular acidifications occurring during inflammation and their expression is increased in rat sensory neurons (particularly ASIC3) in this pathological condition [
10]. Thus, in an attempt to characterize the mechanism through which the BM-21 exerts its antinociceptive action we evaluated BM-21 and thalassiolin B on ASIC currents.
We found that both BM-21 and thalassiolin B were able to decrease the peak amplitude of ASIC currents; indicating that, most probably, the effect of BM-21 on ASIC currents may be because of the presence of thalassiolin B. The maximum effect of BM-21 and thalassiolin B on ASICs currents was about 56% and 30%. Therefore the activity of thalassiolin B could not account for the overall activity of the extract, which suggests that other components are also able to act on the ASIC currents. The presence of other molecules in the extract and in particular of thalassiolins A and C could contribute to this synergic effect [
18].
The thalassiolin B inhibitory action on ASIC currents in DRG neurons was selective, acting only in those neurons with a fast desensitization time-course, without significant effect on those currents with a slow desensitizing time-course, the so-called "ASIC2-like" in DRG neurons [
9], indicating that its effect could be specific. However, because H
+-gated currents in DRG neurons are likely to be caused by the combination of two or more ASIC subunits coassembled as heteromultimers, with a coexistence of multiple channel populations within the same cell [
36,
37], it is not possible to ascertain its selectivity.
Thalassiolin B acts in the same range of potency in DRG neurons (IC
50 = 27.3 ± 2.6 μM) as A-317567 and amiloride. Both produce a concentration-dependent inhibition of ASIC currents with an IC
50 ranging between 2 and 29 μM and 30 and 51 μM in acutely dissociated DRG neurons [
9]. Their inhibitory action is similar to that of streptomycin (32 ± 2.7 μM), neomycin (44 ± 2.6 μM) [
20], amiloride (30 to 51 μM), and A-317567 (2 to 29 μM) [
9]. In contrast, APETx-2 (from the sea anemone
Anthopleura elegantissima) in primary-cultured sensory neurons inhibits ASIC3-like currents with an IC
50 of 216 nM [
38], whereas psalmotoxin 1 (PcTx1, from the tarantula
Psalmopoeus cambridgei) seems to be the most potent and inhibits a subpopulation of H
+-gated currents (ASIC1a-like) in DRG neurons with an IC
50 = 0.7 nM [
39]. Similar to PcTx1 and APETx2 and in contrast with low-molecular-weight compounds of nonpeptidic nature, amiloride and A-317467 [
9], thalassiolin B shows an effect that depends on the application schedule, and it needs to be applied a few seconds before the pH change, suggesting a complex interaction with the ASICs.
For its mechanism of action, thalassiolin B has a selective action among the different ASIC channel subunits sparing those currents with a τ ≥ 400 ms. The pH current curve shows that thalassiolin B did not modify the proton gating of the ASICs in DRG neurons. The steady-state desensitization curves also indicate that its action is not modified by channel desensitization. The effect of thalassiolin B shows no dependence on the pH used to activate the ASIC current, thus indicating that no conformational changes of the molecule as a function of pH are taking place. Interestingly, the coapplication of amiloride occluded the effect of thalassiolin B thus suggesting that they act through a similar mechanism or that the action of amiloride is downstream from thalassiolin B. Our data shows no voltage dependence of its effect indicating that most probably it is not entering into the membrane field potential. It requires preapplication to have a consistent inhibitory effect and it is able only to partially block the ASIC currents (about 30% at its highest concentrations), thus suggesting that thalassiolin B is not an ASIC-pore blocker. Future studies using heterologous expression of ASIC subunits would contribute to define the selectivity and action mechanism of thalassiolin B on the ASICs.
Conclusions
The results of this study demonstrate the antinociceptive action of the T. testudinum extract, BM-21, after a single oral administration in classical thermal- and chemical caused pain in mice. The antinociceptive effects of BM-21 may be partially caused by its antagonism to ASIC channels. Thalassiolin B, the major phenolic constituent isolated from BM-21 also had an antinociceptive behavior in the formalin test and inhibited ASIC currents in DRG neurons indicating that this sulphated flavone glycoside may be responsible, at least in part, for the effects found with BM-21.
To our knowledge, this is the first report of the presence of an ASIC inhibitor in a marine-plant extract and associated to an identified phenolic compound. In comparison with other natural substances of peptidic nature with antinociceptive action such as PcTx1 and APETx2, the nonpeptidic nature of thalassiolin B may have an advantage for future therapeutic applications in the control of pain. However, further studies are still required to ascertain its selectivity within ASIC subunits, and to determine its potency, the time required to attain therapeutic levels in vivo, and its mechanism of action.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
AG, RM, TG, RAM made the behavioral studies on mice. AG, ES, OL and ESoto made the voltage clamp experiments including processing of the data. AL, OPT, ELR did the isolation and characterization of BM-21 and of thalassiolin B. AG and Esoto coordinated and helped with the writing of manuscript. All authors read and approved the final manuscript.